A Critical Role for Eosinophils in Allergic Airways Remodeling

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Science  17 Sep 2004:
Vol. 305, Issue 5691, pp. 1776-1779
DOI: 10.1126/science.1100283


Features of chronic asthma include airway hyperresponsiveness, inflammatory infiltrates, and structural changes in the airways, termed remodeling. The contribution of eosinophils, cells associated with asthma and allergy, remains to be established. We show that in mice with a total ablation of the eosinophil lineage, increases in airway hyperresponsiveness and mucus secretion were similar to those observed in wild-type mice, but eosinophil-deficient mice were significantly protected from peribronchiolar collagen deposition and increases in airway smooth muscle. These data suggest that eosinophils contribute substantially to airway remodeling but are not obligatory for allergen-induced lung dysfunction, and support an important role for eosinophil-targeted therapies in chronic asthma.

Since its discovery by Paul Erlich in 1879, there has been a wealth of information documenting the association between eosinophils and parasitic or allergic diseases (1). The role of eosinophils in allergic disease remains controversial. Although T helper cell 2 (TH2) lymphocytes are thought to drive asthmatic responses, increasing evidence suggests that eosinophils are associated with development of lung dysfunction and subsequent immunopathology (24).

Asthma is a chronic disease characterized by airway hyperresponsiveness (AHR), airway inflammation, and reversible airway obstruction. In addition, structural changes in the airway, termed remodeling, occur as a result of an imbalance in tissue regeneration and repair mechanisms (5, 6). Subepithelial fibrosis is a distinctive feature of airway remodeling and contributes to the thickened airway walls due to the deposition of collagen types I, III, and IV, fibronectin, and other extracellular matrix (ECM) proteins such as tenascin and laminin (7, 8). Increased airway smooth muscle (ASM) mass and excessive mucus secretion from hyperplastic goblet cells are also features of airway remodeling (9, 10).

To define the role of eosinophils in asthma pathophysiology, we used the recently described eosinophil lineage–ablated line, Δdbl GATA mice (11). Deletion of a highaffinity GATA site in the GATA-1 promotor results in a complete ablation of the eosinophil lineage without affecting the development of the other GATA-1–dependent lineages (erythroid, megakaryocytic, and mast cell) (11).

We examined the extent of this mutation on eosinophil recruitment following acute and chronic allergen challenge in a murine model of allergic airways disease (12, 13). Histological examination confirmed that sham-treated Δdbl GATA mice were completely devoid of eosinophils and that allergen challenge failed to induce eosinophilia in the airways and bone marrow of Δdbl GATA mice (Fig. 1, A to D). Eosinophil peroxidase (EPO) analysis (13) of lung tissue (fig S2) and bone marrow (Fig. 1E) confirmed the absence of eosinophils in these tissues. During acute and chronic phases, wild-type (WT) mice showed significant increases in pulmonary eosinophils and lymphocytes. Allergen challenge induced similar numbers of alveolar macrophages and lymphocytes in both WT and Δdbl GATA mice, confirming that the Δdbl GATA mutation was selective for eosinophils [bronchoalveolar lavage (BAL) cell counts and lung EPO are shown in figs. S1 and S2)]. Given the role of GATA-1 in mast cell differentiation (14), histological analysis of chloroacetate esterase–stained tissue sections demonstrated that this mutation had no effect on mast cell numbers (15).

Fig. 1.

Acute allergen challenge induces eosinophil accumulation in the lung and bone marrow of WT (A and C) but not Δdbl GATA mice (B and D). Original magnification, ×20 μm. (E) EPO analysis of bone marrow confirmed that Δdbl GATA mice (solid bars) are devoid of eosinophils compared to WT controls (open bars) before (sham) and after (OA) allergen challenge. Results are means ± SEM (Sham, n = 4 mice; OA, n = 5 mice). Significant differences between respective sham-treated and sensitized/challenged WT or Δdbl GATA mice are indicated as *P < 0.03 and **P < 0.008.

Airway function was assessed using whole-body plethysmography (13). Pulmonary conductance (GL) and compliance (Cdyn) (16), and Penh [a calculated value that correlates with measurement of airway resistance, obstruction, and intrapleural pressure in the same mouse (17)] were assessed on day 25 after acute challenge on days 21 to 24. WT allergen-challenged mice developed a significantly enhanced response to methacholine (Mch) when compared to WT sham-treated animals. In the absence of eosinophils, allergen-challenged Δdbl GATA mice displayed enhanced responses to Mch relative to baseline sham controls that were comparable to those displayed by challenged WT mice (Fig. 2, A and B). Similarly, the Penh responses of ovalbumin (OA)-challenged Δdbl GATA mice to Mch were almost identical to those of their WT-challenged counterparts (fig. S3). During the chronic phase, mice were assessed for changes in lung function weekly until day 55. Although the enhancement following allergen challenge was lower than that seen during the acute phase (at day 25), allergen-challenged WT and Δdbl GATA mice displayed similar enhanced responses to cholinergic stimulation relative to baseline sham controls (fig. S4). Thus, eosinophil deficiency conferred no protection against Mch-induced AHR (during acute and chronic allergen challenge), suggesting that eosinophils are not obligatory for allergen-induced changes in airway physiology.

Fig. 2.

AHR following acute allergen challenge. Sham-treated (dashed lines) WT (◯) or Δdbl GATA mice (⚫) mice were exposed to aerosolized saline, and OA-sensitized (solid lines) WT (▢) and Δdbl GATA mice (◼) were exposed to aerosolized OA on days 21 to 24. About 21 to 24 hours after the last aerosol challenge, mice were anesthetized, intubated, and mechanically ventilated, and airway responses to increasing concentrations of intravenous Mch were assessed. The dose-response curves for (A) pulmonary conductance (GL) and (B) pulmonary compliance (Cdyn) are shown. Results are means ± SEM (Sham, n = 4 mice; OA, n = 8 or 9 mice) of the percentage minimal decrease in pulmonary conductance or compliance obtained after Mch challenge compared with the baseline value just before challenge. Significant differences between respective sham-treated and sensitized/challenged WT or Δdbl GATA mice are indicated as *P < 0.05 to P < 0.01.

Given the role of TH2 cells in the allergic response, we examined TH2 cytokine expression in the lungs of acute and chronically challenged WT and Δdbl GATA mice. TH2 responses in Δdbl GATA mice appeared normal and were similar to those of their WT littermates. Δdbl GATA mice displayed increased BAL and lung interleukin-4 (IL-4), IL-5, and IL-13 protein following acute allergen challenge. Likewise, IL-4 and IL-5 expression during the chronic phase were comparable for WT and Δdbl GATA mice (fig. S5). Numbers of TH2 cells, determined by staining lungs for the TH2 surface marker T1/ST2, were found to be comparable between WT and Δdbl GATA mice (15). Moreover, serum-specific OA–immunoglobulin E was similar (acute OA WT = 3713 ± 539 ng/ml versus OA Δdbl GATA mice = 4059 ± 789 ng/ml; chronic OA WT = 5204 ± 716 ng/ml versus OA Δdbl GATA mice = 5635 ± 741 ng/ml; n = 6 to 9 mice). These data demonstrate that allergen-driven TH2 responses develop in the absence of eosinophils.

TH2 cytokines (IL-4, IL-5, IL-9, and IL-13) and transforming growth factor–β (TGF-β) have been shown to induce subepithelial fibrosis (1824). Recent reports support a potential role for eosinophils in the development of airway remodeling (24). Increased eosinophils in the bronchial mucosa of severe asthmatics have been associated with basement membrane thickening (25), and eosinophils are capable of secreting an array of profibrotic mediators (22, 23). However, studies in which IL-5 activity was inhibited, although associated with a decrease in eosinophil numbers, could theoretically be operating on a number of pathways independent of the eosinophil (24). In light of this ambiguity, we examined the effects of specific eosinophil deficiency on airway remodeling following chronic challenge.

Increased mucus secretion from hyperplastic goblet cells, shown by periodic acid-Schiff (PAS)–positive cells in the bronchial epithelium (13), was similar in WT and Δdbl GATA mice compared to sham controls after acute challenge, and these increases were sustained throughout chronic challenge (fig. S6). Thus, enhanced mucus secretion occurs in allergic airways independent of eosinophils.

Increased subepithelial deposition of ECM proteins, specifically collagen, is a prominent feature of airway remodeling. We examined matrix deposition (collagen and fibrin) in lung sections stained with Martius scarlet blue (MSB) (13, 26). Sham mice showed a thin uniform layer of matrix in peribronchiolar subepithelial regions (Fig. 3, A and B), whereas acute challenge marginally increased fine matrix in both WT and Δdbl GATA mice within some infiltrates (15). Prolonged challenge of WT mice significantly increased matrix deposition in the subepithelial layer of the bronchioles and perivascular regions. Dense fibrils were seen in the subepithelial and submucosal areas and in between the inflammatory cells. In marked contrast, matrix deposition in these same regions was consistently reduced in Δdbl GATA mice when compared with that in WT mice (Fig. 3, A to D; fig. S7, A to D). Quantitative image analysis of MSB-stained lung sections and biochemical measurement of total collagen in lung tissue (13) confirmed that prolonged allergen challenge of WT mice provoked a marked increase (up to threefold) in matrix deposition, as compared with that seen in sham mice, and levels were significantly reduced in challenged Δdbl GATA mice (Fig. 3, E and F, respectively). These results conclusively demonstrate that eosinophils contribute to allergen-induced subepithelial collagen deposition.

Fig. 3.

In the absence of allergen challenge, WT (A) and Δdbl GATA (B) mice exhibited minimal subepithelial MSB staining (blue). In contrast, chronic OA challenge induced a significant increase in MSB staining (C), which was markedly reduced in challenged Δdbl GATA mice (D). Data are representative of 8 to 12 mice per group; original magnifications, ×40. (E) Image analysis of MSB-stained lung sections from sham or chronically challenged WT (open bars) and Δdbl GATA mice (solid bars) confirmed that challenged Δdbl GATA mice were significantly protected from collagen deposition (**P < 0.0001). Results are means ± SEM (n = 8 to 12 mice per group). Significance between Sham WT and OA WT is indicated (*P < 0.0001). (F) Lung collagen was measured in sham and chronically challenged Δdbl GATA mice. Individual values and means (solid lines) for each group are shown (n = 10 to 16 mice per group). Significant differences between Sham WT and OA WT, and between OA WT and Δdbl GATA mice, are indicated as *P < 0.001 and **P < 0.003, respectively.

The effects of eosinophil deficiency on ASM hyperplasia and proliferation were determined by counting the numbers of total and proliferating cell nuclear antigen (PCNA)–positive smooth muscle cells along the basement membrane of three or four bronchioles per animal (13). Prolonged allergen challenge of WT mice induced a significant increase in the total and proliferating number of ASM cells compared with that seen in sham mice. This increase was absent from airways of chronically challenged Δdbl GATA mice (Fig. 4 and fig. S8). Prolonged allergen challenge induces phenotypic changes in ASM cells (27), which could conceivably induce secretion of a number of growth factors, like TGF-β, which contribute to ECM formation. We investigated expression of active TGF-β 1 in WT and Δdbl GATA mice and consistently found no differences in TGF-β1 expression between chronically challenged WT and Δdbl GATA mice (either protein or mRNA). These data suggest that reduced sub-epithelial fibrosis in our model is independent of TGF-β1 expression.

Fig. 4.

Eosinophil deficiency protects against increases in ASM. (A) Number of ASM cells (round and elongated) and (B) proliferating (PCNA-positive) ASM cells along the basement membrane of three or four bronchioles per mouse were determined in sham and chronically challenged WT (open bars) and Δdbl GATA mice (solid bars). Results are means ± SEM for each group (Sham, n = 4 mice; OA, n = 6 mice). Significant difference between OA WT and Δdbl GATA mice is indicated (*P < 0.004).

Our work contrasts with a recent report demonstrating that IL-5–deficient mice are protected from collagen deposition because of a reduction in TGF-β–positive eosinophils (4). We have previously shown that mononuclear cells, presumably macrophages, and not eosinophils are the main secretors of TGF-β1 protein during chronic challenge (12). The reason for this disparity is unclear (4), but variability in protocols may account for the differences seen in the cell source and expression levels of TGF-β. A number of other factors have been demonstrated to be profibrotic in the lung, notably the chemokine MCP-1, thrombin, endothelin-1, and plasminogen activator inhibitor 1 (28). It is difficult to link the presence of these factors to eosinophils specifically. However, the cysteinyl leukotrienes have been shown to be linked to both profibrotic remodeling responses and eosinophils (29, 30). In fact, the eosinophil may be a major source of leukotrienes, often overlooked.

Of importance is that these animal studies are in accordance with observations made in humans. Mild asthmatic patients pretreated with IL-5–specific antibody exhibited significant reduction in tenascin, lumican, and procollagen III (3). Our results independently demonstrate that eosinophils are in part responsible for both collagen and smooth muscle changes in a chronic model of asthma. Further, although the contribution of eosinophils to lung dysfunction has been controversial, we show here that eosinophils are not obligatory for airway physiology changes associated with this disease. Taken together, these data provide a rationale for anti-eosinophil–based therapeutics in chronic allergic airways disease.

Supporting Online Material

Materials and Methods

Figs. S1 to S8


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